Multiple modes for gatekeeping at fungal cell-to-cell channels

Summary

Cell-to-cell channels appear to be indispensable for successful multicellular organization and arose independently in animals, plants and fungi. Most of the fungi obtain nutrients from the environment by growing in an exploratory and invasive manner, and this ability depends on multicellular filaments known as hyphae. These cells grow by tip extension and can be divided into compartments by cell walls that typically retain a central pore that allows intercellular transport and cooperation. In the major clade of filamentous Ascomycota, integrity of this coenocytic organization is maintained by Woronin body organelles, which function as emergency patches of septal pores. In this issue of Molecular Microbiology, Bleichrodt and co-workers show that Woronin bodies can also form tight reversible associations with the pore and further link this to variation in levels of compartmental gene expression. These data define an additional modality of Woronin body-dependent gatekeeping. This commentary focuses on the implications of this work and the potential role of different modes of pore gating in controlling the growth and development of fungal tissues.

Cells in multicellular tissues must communicate in order to co-ordinate their development and physiological functions. This generally takes two forms: it can occur through signalling molecules that are released by one cell and perceived through receptors in others, or it can occur through channels that link the cytoplasm of adjacent cells. The latter form of communication allows cells to couple and co-ordinate their activities, and animals, plants and fungi each possess different kinds of channels that serve this function. Animal gap junctions (Goodenough and Paul, 2009) and plant plasmodesmata (Xu and Jackson, 2010) have been well studied and each plays roles in co-ordinating systemic cellular behaviour and development. By comparison, less is known about the composition and function of fungal cell-to-cell channels (septal pores).

Most of the fungi grow through tip extension of cellular filaments called hyphae. In early diverging phyla, such as Mucoromycotina, hyphae are rarely compartmentalized by cell walls. By contrast, in later diverging filamentous Ascomycota and Basidiomycota, tip growing cells periodically produce septal walls that compartmentalize the hypha in their wake (Stajich et al., 2009; Jedd, 2011). Ultimately, the growth and branching of hyphae produces a radially organized colony in which the periphery is enriched in tip growing apical cells, while increasingly central regions of the colony contain older compartments (Fig. 1A). Septa typically retain a central pore that allows direct communication and transport between adjacent compartments (Fig. 1B), which, in some species, can extend to the tip-directed bulk flow of protoplasm (Lew, 2005). If hyphae are severed to uncouple apical from subapical compartments, the growth rate of apical compartments is instantly diminished (Trinci, 1973). However, at a defined distance from the growth front, severing apical from subapical compartments does not affect tip growth. The region of the colony where compartments contribute to tip growth is known as the peripheral growth zone (Fig. 1A and B). It varies in size from species to species, but typically comprises dozens of compartments (Trinci, 1973). In mature colonies, this type of cooperation can lead to remarkable rates of tip growth, which can approach 1 μm s⁻¹.

Figure 1. Radial organization and compartmental differentiation in a fungal colony.

A. A typical colony is characterized by rapid tip growth at the leading edge. The peripheral growth zone, shown in green, is the region where subapical compartments contribute to polarized growth of the apical cell. Regions at the interior of the colony, shown in red, typically differentiate to participate in asexual spore production and sexual development. The colony schematic is adapted from A. H. R. Buller's Researches on Fungi. For simplicity septa are not depicted.

B. Septation occurs periodically in the apical compartment where Woronin bodies (shown in green) are also produced. These associate with new septa where they are retained, probably through cortex tethering. A second type of pore plug, shown in red, has been associated with older regions of the colony, but its function in promoting differentiation has yet to be established. Bleichrodt et al. (2012) show that compartmental variation in the level of gene expression within the peripheral growth zone depends on Woronin bodies, suggesting an additional gatekeeping function that promotes differentiation (shown in varying shades of blue).

C. When the hypha is ruptured, Woronin bodies are poised to plug the pore, resist intracellular turgor (see the bowed septum) and promote membrane resealing and new tip production.

Cellular coupling through such large cell-to-cell channels is especially advantageous for invasive and foraging growth, but also presents the risk of catastrophic cell death in the event of cell lysis. In the filamentous Ascomycota (Pezizomycotina), pore-associated organelles known as Woronin bodies ameliorate this risk (Markham and Collinge, 1987). These are centred on a crystalline core of the matrix protein HEX (Jedd and Chua, 2000) and bud from peroxisomes to constitute a physically and functionally distinct subcompartment (Liu et al., 2008; 2011). When a hyphal compartment is ruptured, the Woronin body crystalline core plugs the pore to stem the loss of protoplasm (Yuan et al., 2003). This is accompanied by membrane sealing over the pore and the resumption of polarized growth (Fig. 1C). The emergency patch function appears to be a conserved aspect of Woronin body function as uncontrolled protoplasmic bleeding has been observed in all hex mutants examined to date (Jedd and Chua, 2000; Tenney et al., 2000; Soundararajan et al., 2004; Maruyama et al., 2005). However, additional functions have not been ruled out, and the Woronin body has been linked to cellular redox regulation (Kim et al., 2009), microtubule organization (Zekert et al., 2010) and development (Soundararajan et al., 2004; Engh et al., 2007).

In this issue, Bleichrodt et al. (2012) provide new insights into Woronin body function that raise interesting questions about the regulation of intercompartmental transport. They employ laser ablation to puncture apical compartments of Aspergillus oryzae colonies and observe the degree to which adjacent compartments release protoplasm through the wound proximal septal pore. Two general types of responses are observed. In about 60% of the cases, cytoplasm streams briefly and then ceases, and these are referred to as open pores. In the remainder, there is no cytoplasmic release, and these are referred to as closed pores. Sequential ablation of the first, second and third compartments further indicates that the open and closed states are independently determined. The authors next found that all pores are open when the hex gene is deleted, indicating that the open and closed states reflect differences in Woronin body status.

The authors note that given these results, an apical compartment has roughly a 5% probability of sharing a continuous cytoplasm with the seventh compartment. How can these results be reconciled with the peripheral growth zone, which presumably functions through intercellular transport? One possibility is that the open and closed states are highly dynamic and in this case, significant cytoplasmic continuity could be achieved even with a high rate of instantaneous closure. In addition, while bulk trafficking of protoplasm has been observed in some species (Lew, 2005), in others, intercellular transport may be more selective. Moreover, it remains possible that at a closed septum, a gap exists between the Woronin body and pore that still permits trafficking of growth-associated factors such as small secretory vesicles. In this context, it is also worth noting that the septum possesses a microtubule-organizing centre (sMTOC) (Zekert et al., 2010), which may promote such intercompartmental transport.

What could be the molecular basis for dynamic regulation of Woronin body positioning? Transmission electron microscopy (TEM) shows that Woronin bodies are typically tethered at a distance of 50 to 100 nm from the pore and several are found on both sides of the septum (Momany et al., 2002). Laser tweezers have been used to pull Woronin bodies away from the septum and these recoil and return to their original position upon release, suggesting an elastic tether (Berns et al., 1992). In Aspergillus, the molecular basis of tethering remains to be determined. However, in Neurospora crassa, the Leashin protein functions in Woronin body inheritance and tethering by promoting cell cortex attachment (Ng et al., 2009). Leashin homologues are found in other filamentous Ascomycetes, and these are sufficiently large to account for the spacing between the Woronin body and pore (Ng et al., 2009). Regulation at the level of Leashin or its associates could therefore account for the septum-to-septum variation observed by Bleichrodt et al. (2012). In addition, the cytoplasmic SOFT protein, which functions to promote cell–cell fusion, has been shown to reversibly associate with pores in a stress-dependent manner (Maruyama et al., 2010). This indicates that composition of the pore is dynamic and provides another potential mechanism for producing variation at the pore. It is also possible that Woronin bodies are not systematically pore-associated and this could also account for septum-to-septum variation. The use of functional Woronin body GFP markers can help to distinguish these different possibilities.

Other outstanding questions concern phyla that possess different mechanisms for dealing with pore gating and cellular wound healing. For example, in filamentous Basidiomycota (Agaricomycotina), the endoplasmic reticulum-derived septal pore cap (SPC) gates septal pores (van Peer et al., 2010), and in this system, apical septa are also found in open and closed states (van Peer et al., 2009). In this case, the link between gating and the SPC remains to be investigated. It is also worth studying the early diverging fungi, which produce extensive hyphal networks that are generally not afforded protection by septal walls. In these systems, cross-linking of protoplasm near the point of hyphal damage appears to limit the loss of protoplasm and initiates the deposition of new plasma membrane (T. A. Nguyen and G. Jedd, unpubl. obs.). Understanding how this process is triggered and terminated also awaits future research.

Apical and subapical regions of the colony express different suites of genes to support distinct developmental and physiological programmes (Tey et al., 2005; Masai et al., 2006; Levin et al., 2007; Kasuga and Glass, 2008). Surprisingly, significant variation has been observed in gene expression between neighbouring hyphae in apical regions of the colony (de Bekker et al., 2011). For example, in Aspergillus niger, adjacent apical hyphae can express different levels of genes encoding secreted proteins (Vinck et al., 2005; 2011), and this appears to reflect two classes of hyphae with different levels of transcriptional/translational activity (Vinck et al., 2011). Interestingly, Bleichrodt et al. (2012) find that deletion of hex also diminished this form of differential gene expression. Together, these data suggest an additional mode for Woronin body gatekeeping that restricts intercompartmental communication and thus promotes differences in cellular metabolism. Understanding which factors underlie this compartmental variation and its physiological function remains challenges for future work.

Woronin bodies are not the only means of pore gating. A second system, originally identified by TEM, consists of electron-dense aggregates that line and occlude the septal pore. These structures are not membrane-delimited and appear to be in direct contact with the cytoplasm (Furtado, 1971; Trinci and Collinge, 1973; Rosing, 1981; Read and Beckett, 1996). Interestingly, this form of plugging appears to occur increasingly with age (Trinci and Collinge, 1973), suggesting association with the peripheral growth zone. Recent work has identified dozens of cytosol-based intrinsically disordered proteins that localize to the pore. Some of these have the inherent tendency to aggregate and their pore association increases under conditions of cell stress and during compartmental cell death (Lai et al., 2012). Together, these results suggest that cellular physiology controls this second form of pore occlusion. However, its interplay with the Woronin body and potential role in developmental patterning has yet to be investigated. Continued use of mutants and laser ablation experiments like those employed by Bleichrodt et al. (2012) can help determine how these diverse systems of pore closure are co-ordinated, as well as their potential role in physiological and developmental patterning.

Acknowledgements

We thank Rachel Loh for critical reading. The Temasek Life Sciences Laboratory and Singapore Millennium Foundation support research in the Jedd group.